Rhabdoviruses and Tospoviruses February 12, 2003 VSV: A

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Pl Pa 606 Rhabdoviruses and Tospoviruses February
12, 2003
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Enveloped Viruses
Capsid packaging genome surrounded by
lipid-containing membrane
Nucleocapsid
Animal Viruses Icosahedral (Herpes viruses,
Togaviruses) or Helical (Influenza viruses,
Rhabdoviruses)
expanded or loose structure
Membrane Envelope
Bilayer membrane structure derived from host
cell membrane
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VSV A Typical Enveloped Virus
Proteins Viral encoded
Envelope proteins Integral transmembrane (gp
Spikes) or Peripheral (Matrix)
Lipids Determined by host (acquired by budding)
Composition depends on which host, which membrane
From Flint et al. Pinciples of Virology (2000),
ASM Press
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Targeting of virus glycoproteins is key for
enveloped viruses

• Glycoproteins must be correctly processed in the
  ER and Golgi complex glycosylation
• Brefeldin A will invariably block infection of
  enveloped viruses, but will not affect genome
  replication (compare with poliovirus)
• The glycoprotein is targeted to a specific
  cellular membrane ER, Golgi, Plasma Membrane
• retention motifs for ER/Golgi (short amino acid
  sequences)
• Determines site of enveloped virus assembly
  (buddingl)

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Most Animal Viruses Bud at the Plasma Membrane
The Example of VSV, a Rhabdovirus
The localization of viral GPs to specific
cellular membranes determines sites of assembly
and budding.
G protein processed via secretory pathway
Assembly at plasma membrane sites where G inserted
(Host proteins excluded)
From Flint et al. Pinciples of Virology (2000),
ASM Press
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Some Enveloped Animal Viruses Bud at Internal
Membranes

• For viruses that bud into the secretory pathway,
  the virions will traverse
• the remainder of the pathway to be released.

Flint et al. (2000) Principles of Virology, ASM
Press
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Enveloped Plant Viruses

• Tomato Spotted Wilt Virus (TSWV) a Bunyavirus

Animal bunyaviruses replicate in the cytoplasm.
Initially bud at Golgi membranes virions in
Golgi-derived vesicles fuse at plasma membrane to
release virus particles.

• Sonchus Yellow Net Virus (SYNV) and Lettuce
  Necrotic Yellows Virus
• Rhabdoviruses

Animal rhabdoviruses replicate in the cytoplasm
and bud at plasma membrane

• From which cellular membranes do plant viruses
  acquire
• their envelopes?

• Why would a plant virus need an envelope?

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Enveloped Plant Viruses Acquire Their Envelopes
from Internal Cell Membranes

• Tomato Spotted Wilt Virus (TSWV) is a Bunyavirus

Bunyaviruses replicate in the cytoplasm and bud
at Golgi membranes

• Sonchus Yellow Net Virus (SYNV) and Lettuce
  Necrotic Yellows Virus
• (LNYV) are Rhabdoviruses

Animal rhabdoviruses replicate in the cytoplasm
and bud at plasma membrane
LNYV (a cytorhabdovirus) Cytoplasmic replication
like VSV
Envelope apparently acquired from outer nuclear
envelope (continuous with ER)
Virus accumulates in cytoplasmic vesicles
SYNV (a nucleorhabdovirus) Replicates in the
nucleus!
Envelope acquired from inner nuclear membrane
Virions accumulate in perinuclear space
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Rhabdoviruses
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Plant Rhabdoviruses
Genus in Rhabdoviridae 75 viruses (uncertain
all are!)
Most members assigned based on virion morphology,
cursory infectivity studies and crude physical
characterization of virus particles. Only 15
definitely assigned as nucleorhabdo- or
cytorhabdoviruses
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Genome Organization of Plant and Animal
Rhabdoviruses
SYNV a plant rhabdovirus
VSV an animal rhabdovirus
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SYNV-Encoded Proteins
Some biochemical and structural evidence, much by
inference from VSV
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Plant Rhabdoviruses Solve the Cell Wall Problem
by Budding at Internal Membranes
Nucleorhabdovirus (SYNV)
Cytorhabdovirus (LNYV)
Jackson et al (1999). inEncycl. of Virology
31531.
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VSV Nonsegmented RNA Strategy
Transcription monocistronic mRNAs
Pol stops and restarts at ig junctions

• N regulates switch from transcription to
  replication of full-length genome

Low N mRNA synthesis favored (not encapsidated)
High N Nascent RNA encapsidated, L reads
through intergenic junctions
From Flint et al. Principles of Virology (1999),
ASM Press
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VSV Termination and Reinitiation at Intergenic
Regions

• Pol (PL) initiates synthesis at 3 end

• Leader RNA made, synthesis stops in ig

• RNA synthesis reinitiates at 3 end of N

• Reinitiation at 3 of P

Start-Stop continues until all 5 mRNAs made
From Flint et al. Principles of Virology (1999),
ASM Press
Reinitiation does not occur after L mRNA made
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Tospoviruses
Genus in Bunyaviridae
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Bunyaviruses
The plant Bunyavirus (Tospovirus) genome is
comprised of 3 segments organized like that of
the animal Bunyaviruses except for the M (medium
size) segment, which encodes the 2 glycoproteins
G1 and G2.
The Tospovirus M segment is uniquely ambisense to
encode an extra protein NSm the movement
protein!
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Tospovirus-Encoded Proteins

• Functions supported by mutational, biochemical
  and cell-based studies.

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Tospovirus Genome is Unique Among Bunyaviruses in
its M Segment
M segment encodes gycoproteins G1 G2
Animal-infecting viruses

• Strictly negative sense strategy

• Single ORF, co-translational cleavage)

• NSM of bunya and phlebo 11-14 kD

Tospoviruses

• Uniquely ambisense

• Subgenomic RNA encodes NSM of 34 kD Movement
  Protein

from Fields et al., Virology, 3rd edition
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Tospoviruses (Plant Bunyaviruses) Use the Return
Pathway from the Golgi to the ER
Animal Bunyaviruses bud into the Golgi and
transit in secretory vesicles to be released at
the plasma membrane
Lazarowitz (2001) in Fields Virology, 4th
Ed.,Lippincott
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Why Does a Virus Need an Envelope When Infecting
a Plant?
It doesnt!
RNPs NO enveloped particles
anti-N react
anti-G1, anti-G2 not react
Conclusion Enveloped virions and GPs needed for
transmission by and life cycle in thrips
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What is the Function of the Envelope and
Glycoproteins for a Plant Virus?

• Infection of the Insect Vector

• Tospoviruses and Rhabdoviruses infect and
  multiply
• in their insect vectors

• Viral-encoded glycoproteins and envelope needed
• for their animal virus life cycles

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Bunyavirus Transcription Cap Snatching
Primary transcription of complementary mRNAs from
negative-sense vRNA
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Evidence for Host Cell Primers on Bunyavirus
Transcripts
TSWVAlMV infected cells AlMV sequences found
at 5-ends of TSWV mRNAs
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Bunyavirus Transcription
Bunyavirus transcription differs from Influenza
Virus
NO spliced transcripts
Inferred primers derived from host cytoplasmic
mRNAs
TSWV AlMV co-infection suggests cap snatching
does occur in cytoplasm
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Technical Advances for Investigating TSWV
Kikkert et al. J. Gen. Virol. 781755 (1997)
Tobacco protoplast system for TSWV multiplication
Nagata et al. Virus Res. 4959 (1997) Thrips
cell culture for TSWV multiplication
Qiu et al. Virol. 244186 (1998) Genome
Reassortment studies based on identified RFLPs in
RT-PCR amplified fragments of 3 TSWV strains
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Supplement Transcription and Replication of
Tospovirus 3 Genomic Segments
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TSWV Transcription and Replication
snatched cap